Cytoplasmic Calcium Levels Research Articles

Poliovirus protein 2BC increases cytosolic free calcium concentrations.

Poliovirus-infected cells undergo an increase in cytoplasmic calcium concentrations from the 4th h postinfection. The protein responsible for this effect was identified by the expression of different poliovirus nonstructural proteins in HeLa cells by using a recombinant vaccinia virus system. Synthesis of protein 2BC enhances cytoplasmic calcium concentrations in a manner similar to that observed in poliovirus-infected cells. To identify the regions in 2BC involved in modifying cytoplasmic calcium levels, several 2BC variants were generated. Regions present in both 2B and 2C are necessary to augment cellular free calcium levels. Therefore, in addition to inducing proliferation of membranous vesicles, poliovirus protein 2BC also alters cellular calcium homeostasis.

Overexpression of the rat sarcoplasmic reticulum Ca2+ ATPase gene in the heart of transgenic mice accelerates calcium transients and cardiac relaxation.

The Ca2+ ATPase of the sarcoplasmic reticulum (SERCA2) plays a dominant role in lowering cytoplasmic calcium levels during cardiac relaxation and reduction of its activity has been linked to delayed diastolic relaxation in hypothyroid and failing hearts. To determine the contractile alterations resulting from increased SERCA2 expression, we generated transgenic mice overexpressing a rat SERCA2 transgene. Characterization of a heterozygous transgenic mouse line (CJ5) showed that the amount of SERCA2 mRNA and protein increased 2. 6-fold and 1.2-fold, respectively, relative to control mice. Determination of the relative synthesis rate of SERCA2 protein showed an 82% increase. The mRNA levels of some of the other genes involved in calcium handling, such as the ryanodine receptor and calsequestrin, remained unchanged, but the mRNA levels of phospholamban and Na+/Ca2+ exchanger increased 1.4-fold and 1.8-fold, respectively. The increase in phospholamban or Na+/Ca2+ exchanger mRNAs did not, however, result in changes in protein levels. Functional analysis of calcium handling and contractile parameters in isolated cardiac myocytes indicated that the intracellular calcium decline (t1/2) and myocyte relengthening (t1/2) were accelerated by 23 and 22%, respectively. In addition, the rate of myocyte shortening was also significantly faster. In isolated papillary muscle from SERCA2 transgenic mice, the time to half maximum postrest potentiation was significantly shorter than in negative littermates. Furthermore, cardiac function measured in vivo, demonstrated significantly accelerated contraction and relaxation in SERCA2 transgenic mice that were further augmented in both groups with isoproterenol administration. Similar results were obtained for the contractile performance of myocytes isolated from a separate line (CJ2) of homozygous SERCA2 transgenic mice. Our findings suggest, for the first time, that increased SERCA2 expression is feasible in vivo and results in enhanced calcium transients, myocardial contractility, and relaxation that may have further therapeutic implications.

T cells deficient in inositol 1,4,5-trisphosphate receptor are resistant to apoptosis.

The type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) calcium release channel is present on the endoplasmic reticulum of most cell types. T lymphocytes which have been made deficient in IP3R1 lack detectable IP3-induced intracellular calcium release and exhibit defective signaling via the T-cell receptor (TCR) (T. Jayaraman, E. Ondriasova, K. Ondrias, D. Harnick, and A. R. Marks, Proc. Natl. Acad. Sci. USA 92:6007-6011, 1995). We now show that IP3R1-deficient T cells are resistant to apoptosis induced by dexamethasone, TCR stimulation, ionizing radiation, and Fas. Resistance to TCR-mediated apoptosis in IP3R1-deficient cells is reversed by pharmacologically raising cytoplasmic calcium levels. TCR-mediated apoptosis can be induced in calcium-free media, indicating that extracellular calcium influx is not required. These findings suggest that intracellular calcium release via the IP3R1 is a critical mediator of apoptosis.

Calcium Metabolism in Blood and Peritoneal L Ymphocytes from Continuous Ambulatory Peritoneal Dialysis Patients

Cellular immune function in peritoneal dialysis patients has been shown to be depressed, but the mechanism of this immunosuppression has not been ascertained. Because calcium is an important mediator of lymphocyte activation, this study was designed to investigate if there was an alteration of calcium metabolism in the lymphocytes of continuous ambulatory peritoneal dialysis (CAPD) patients. Sixteen CAPD patients were studied at the initiation of CAPD and after two months of treatment. Twenty-three normal controls were also enrolled in the study. Cytoplasmic calcium changes were investigated in response to the mitogen phytohemagglutinin (PHA) in peripheral blood and peritoneal lymphocytes, using the intracellular calcium probe indo-1 and flow cytometry. Baseline cytoplasmic calcium levels and changes in cytoplasmic calcium in response to PHA were assessed at the initiation of CAPD and after two months of therapy. Peripheral lymphocytes of patients and controls had similar calcium baseline levels, but the peritoneal lymphocytes had baseline cytoplasmic calcium levels averaging 81% higher than the corresponding calcium levels of the patients' peripheral blood lymphocytes. As compared to peripheral lymphocytes, the response to PHA stimulation was significantly less in the peritoneal lymphocytes, increasing an average of only 46.8% above baseline. Peripheral blood lymphocytes of the patients responded by an average increase of 78.9% over baseline. Control cells increased an average of 66.3% over baseline. Follow-up studies done two months after the initiation of CAPD indicated there were no significant changes (as compared to month 0) that occurred in baseline or stimulated intracellular calcium concentrations. While the peripheral lymphocytes of CAPD patients respond adequately to PHA, the high baseline calcium levels of the peritoneal lymphocytes suggest that these cells may be in a state of chronic activation and may respond minimally to an antigenic challenge.

Neurotransmitter- and Growth Factor-Induced cAMP Response Element Binding Protein Phosphorylation in Glial Cell Progenitors: Role of Calcium Ions, Protein Kinase C, and Mitogen-Activated Protein Kinase/Ribosomal S6 Kinase Pathway

To understand how extracellular signals may produce long-term effects in neural cells, we have analyzed the mechanism by which neurotransmitters and growth factors induce phosphorylation of the transcription factor cAMP response element binding protein (CREB) in cortical oligodendrocyte progenitor (OP) cells. Activation of glutamate receptor channels by kainate, as well as stimulation of G-protein-coupled cholinergic receptors by carbachol and tyrosine kinase receptors by basic fibroblast growth factor (bFGF), rapidly leads to mitogen-activated protein kinase (MAPK) phosphorylation and ribosomal S6 kinase (RSK) activation. Kainate and carbachol activation of the MAPK pathway requires extracellular calcium influx and is accompanied by protein kinase C (PKC) induction, with no significant increase in GTP binding to Ras. Conversely, growth factor-stimulated MAPK phosphorylation is independent of extracellular calcium and is accompanied by Ras activation. Both basal and stimulated MAPK activity in OP cells are influenced by cytoplasmic calcium levels, as shown by their sensitivity to the calcium chelator bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid. The kinetics of CREB phosphorylation in response to the various agonists corresponds to that of MAPK activation. Moreover, CREB phosphorylation and MAPK activation are similarly affected by calcium ions. The MEK inhibitor PD 098059, which selectively prevents activation of the MAPK pathway, strongly reduces induction of CREB phosphorylation by kainate, carbachol, bFGF, and the phorbol ester TPA. We propose that in OPs the MAPK/RSK pathway mediates CREB phosphorylation in response to calcium influx, PKC activation, and growth factor stimulation.

Location of endoplasmic reticulum relative to sarcoplasmic reticulum in cultured chick myotubes.

Calcium plays a major role as a second messenger modulating a wide range of cellular processes (1). Endoplasmic and sarcoplasmic reticulum calcium pumps (SERCAs) play a major role in controlling the levels of cytoplasmic calcium but the way in which these pumps are targeted to and maintained in specific cellular compartments is unclear. In skeletal muscle cells it has been assumed that either proteins destined for the sarcoplasmic reticulum (SR) are synthesised on the endoplasmic reticulum (ER) and that the SR forms by extension of the ER membrane or that regions of ER develop into SR (2). However, it is not known what signals result in either proteins moving from ER to SR or in proteins being synthesised on newly forming SR. A similar targeting problem may exist in non-muscle cells where it has been suggested that there is more than one discernible calcium store and that there am. specialised regions of ER involved in calcium uptake and release, i.e.. the calciosomes (3). If there is more than one type of store then there must be mechanisms for targeting calcium pumps to these stores. In order to study this process more closely it is first necessary to determine whether it is possible to discriminate between ER and SR. This has been achieved using antibodies against proteins normally associated with ER and SR in conjunction with laser confocal microscopy. Antibodies against, the ER resident transmembranous protein calnexin (4). the ER retention signal KDEL (5) and the calcium pump (SERCAla) (6) have been used with cultured chick myotubes. Primary cultures of myotubes were prepared from 11-day old embryonic chick breast muscle (modified from (7)). Chopped muscle tissue from 4 birds was dispersed in 10 ml HBSS (calciummagnesium free) supplemented with 0.25% Trypsin-EDTA and stirred for 20 minutes at stirred for 20 minutes at 37C.Undisrupted material was removed following sedimentation and the digestion was stopped by the addition of 10 ml HBSS (calcium-magnesium free) supplemented with soya bean trypsin inhibitor (80 pg ml'), DNAase

Calcium regulation of gene expression: isn't that spatial?

Calcium regulation of gene expression: isn't that spatial?

Improved blood compatibility of drawn polyamide sheets

A series of aliphatic polyamide (nylon) sheets with different draw ratios were prepared and their blood-contacting properties examined in relation to the bulk and surface characteristics. Increases in both the molecular orientation and crystallinity were observed with increasing draw ratio. Their surface wettability slightly decreased with draw ratio, indicating less amide linkages at their surfaces by the drawing process. An increase in contact angle hysteresis by the drawing process is thought to be due to the existence of more amorphous chains at the surfaces. The adsorption of albumin and fibrinogen onto nylon sheets was significantly reduced with draw ratio, suggesting the formation of well-established crystalline-amorphous microdomain structures at the surfaces. Blood compatibility of these surfaces was greatly improved with draw ratio in terms of a change in cytoplasmic calcium levels in platelets contacting these surfaces. Thus, it is suggested that the control of crystalline-amorphous microdomain structures by the drawing process is a feasible approach to improve blood compatibility of commercially available semicrystalline polyamides.

Measuring cytoplasmic calcium level in Citrus protoplasts using the fluorescent probe indo-1

Summary We measured cytoplasmic free calcium in protoplasts of Citrus sinensis Osb. Protoplasts were isolated from cell-suspension cultures grown as microcalli. The cell surface presented acid polysaccharide filaments, probably responsible for cell adhesion. These filaments were disrupted by enzymatic digestion liberating the cells and subsequently the protoplasts. Protoplasts were loaded with the pentapotassium salt of indo-1 by incubation in acidic solutions of this calcium indicator. Loading increased with decreasing pH and osmotica. Dye loaded in this manner was distributed only in the cytoplasm. The protoplast autofluorescence was very low and did not interfere with measurements. Cytoplasmic free-calcium concentration in Citrus protoplasts, using microspectrofluorimetry, was estimated to be 154 nmol·L −1 . This resting value was increased when protoplasts were exposed to high extracellular Ca 2+ (1 mmol · L −1 ) or 6BA (1 μmol · L −1 ) suggesting that the pathway in cytokinin signal transduction in these cells may involve a Ca 2+ uptake facilitation from extracellular medium.

Membrane-proximal calcium transients in stimulated neutrophils detected by total internal reflection fluorescence

A novel fluorescence microscope/laser optical system was developed to measure fast transients of membrane-proximal versus bulk cytoplasmic intracellular calcium levels in cells labeled with a fluorescent calcium indicator. The method is based on the rapid chopping of illumination of the cells between optical configurations for epifluorescence, which excites predominantly the bulk intracellular region, and total internal reflection fluorescence, which excites only the region within approximately 100 nm of the cell-substrate contact. This method was applied to Fluo-3-loaded neutrophils that were activated by the chemoattractant N-formyl-met-leu-phe. Chemoattractant-activated cells showed 1) transient increases in both membrane-proximal and bulk cytosolic Ca2+ that peaked simultaneously; 2) a larger fractional change (20-60%) in membrane-proximal Ca2+ relative to bulk cytosolic Ca2+ that peaked at a time when the main Ca2+ transient was decreasing in both regions and that persisted well after the main transient was over. This method should be applicable to a wide variety of cell types and fluorescent ion indicators in which membrane-proximal ionic transients may be different from those deeper within the cytosol.

Modulation of intercellular adhesion molecule 1 (ICAM-1) expression on the human mast-cell line (HMC)-1 by inflammatory mediators

The effect of inflammatory mediators on the expression of several surface adhesion molecules on the human mast-cell line (HMC)-1 was studied. By flow cytometry, it could be shown that among several surface adhesion molecules (ICAM-1/CD54, VLA-4/CD49d, Mac-1/CD11b, LFA-1/CD11a, LFA-2/CD2, LFA-3/CD58, VCAM-1), only the constitutively expressed immunoglobulin family member intercellular adhesion molecule-1 (ICAM-1) is modulated by proinflammatory cytokines on HMC-1 mast cells. Stimulation with tumor necrosis factor-a (TNF-alpha) and interferon-gamma (IFN-gamma) resulted, in addition to interleukin-(IL-)4, in selective upregulation of ICAM-1 expression. Costimulation of either IL-4 or IFN-gamma with TNF-alpha further increased the ICAM-1 expression as compared to the stimuli alone. In contrast, stem-cell factor (SCF), granulocyte/macrophage colony-stimulating factor (GM-CSF), IL-10, IL-8, monocyte chemotactic and activating factor (MCAF), and the complement split product C5a failed to modulate the expression of any adhesion molecule examined. The levels of cytoplasmic free calcium in HMC-1 mast cells were not altered by cross-linking surface ICAM-1, suggesting linkage of other intracellular signaling pathways. This cytokine-induced upregulation of ICAM-1 expression might reveal a putative regulatory mechanism of mast-cell interaction with effector cells bearing the counterparts of ICAM-1 (CD54), the molecules Mac-1 (CD11b/CD18) and leukosialin (CD43), and the principal ligand LFA-1 (CD11a/CD18).

Transmitter-induced calcium signalling in cultured neurons of the insect brain

The insect brain offers a unique opportunity for cell biological investigation of calcium signalling in relatively homogeneous neuronal populations, such as the mushroom body neurons. This review summarizes fluorescence imaging experiments with the indicator dye fluo-3 to investigate the calcium responses to transmitter stimulation of honeybee mushroom body neurons in primary culture. Application of acetylcholine (ACh) or nicotine promoted a calcium influx into the cell body and neurites. The increase in intracellular calcium after ACh stimulation was blocked by α-bungarotoxin. These results support previous histochemical studies that suggested the expression of nicotinic cholinergic receptors on Kenyon cells. An increase in cytoplasmic calcium levels leads in specific neurons to the generation of nitric oxide (NO) by a Ca 2+/calmodulin activated NO synthase. NO is thought to diffuse as a short-lived messenger molecule through the plasma membrane. Using a sensitive photometric assay it could be shown that dissociated cells from the locust brain release NO after stimulation by agents elevating cytoplasmic Ca 2+ levels and by the excitatory neurotransmitter ACh. The experiments in dissociated culture therefore allow the demonstration of a Ca 2+-dependent release during nerve cell depolarization, which is a basic requirement for identifying NO as a messenger molecule.

Physical and functional association of Fc mu receptor on human natural killer cells with the zeta- and Fc epsilon RI gamma-chains and with src family protein tyrosine kinases.

We recently reported that Fc mu R on NK cells is a signal transducing protein that stimulates a rapid increase in the level of cytoplasmic free calcium upon binding of IgM. This study was designed to examine signal transduction via the Fc mu R on NK cells and to characterize intracellular second messengers activated by IgM. Immunoprecipitation of IgM-bound Fc mu R by IgM-specific Ab coimmunoprecipitated the zeta- and Fc epsilon RI gamma-chains. Furthermore, engagement and clustering of Fc mu R by polyclonal IgM induced tyrosine phosphorylation of the zeta- and Fc epsilon RI gamma-chains, indicating their functional association with the Fc mu R-induced signal transduction cascade. Ligand-induced clustering of the Fc mu R also induced activity of src family kinases, Lck, Fyn, Lyn, and Src, as well as their physical interaction with the receptor. Triggering via Fc mu R also induced the activity of Syk and Zap-70, tyrosine kinases demonstrated to associate with zeta and Lck. Phospholipase C-gamma 1 and phosphatidylinositol 3-kinase were identified as substrates phosphorylated on tyrosine, as down-stream components of the signaling pathway activated in NK cells by polyclonal IgM. Although the Fc mu R on NK cells has not yet been biochemically characterized, our results suggest that the zeta- and Fc epsilon RI gamma-chains are functional subunits of this as well as other important cell surface receptors and that the Fc mu R is coupled either directly or indirectly to nonreceptor tyrosine kinases, which phosphorylate and thereby activate regulatory enzymes such as phospholipase C-gamma 1 and phosphatidylinositol 3-kinase.

G Proteins Regulate Dihydropyridine Binding to Moss Plasma Membranes

The role of calcium as an activator and regulator of many biological processes is linked to the ability of the cell to rapidly change its cytoplasmic calcium levels. Calcium acts as an intracellular messenger in hormone-induced bud formation during the development of the moss Physcomitrella patens. Calcium transport and ligand binding studies have implicated plasma membrane-localized 1, 4-dihydropyridine (DHP)-sensitive calcium channels in this increase in cellular calcium. To understand the regulation of the moss calcium channel, we investigated the involvement of GTP binding regulatory proteins (G proteins). Guanosine 5'-(gamma-thio)triphosphate (GTPgammaS), a nonhydrolyzable GTP analog that locks G proteins into their active state, stimulated DHP binding to high affinity receptors in the moss plasma membrane. DHP binding was measured as the ability of the DHP agonist Bay K8644 or the DHP antagonist nifedipine to compete with the DHP arylazide [3H]azidopine for binding to moss plasma membranes. G protein stimulation of binding was seen when competition was carried out with either nifedipine or Bay K8644. G proteins regulated the rates of association and dissociation of bound [3H]azidopine, and stimulation was dependent on GTPgammaS concentration. Guanosine 5'-(beta-thio)diphosphate, a GDP analog that locks G proteins into their inactivated state, did not affect the dose dependence of either the agonist or the antagonist. These results suggest that G proteins may act via a membrane-delimited pathway to regulate calcium channels in the moss plasma membrane.

Modulation of neutrophil migration by exogenous gaseous nitric oxide.

We studied the effect of exogenous nitric oxide (NO) on migration of rabbit peritoneal neutrophils. Exogenous NO enhanced random migration of neutrophils in a concentration-dependent way. An optimally stimulatory effect was observed with 0.5 microM NO, whereas at higher NO concentrations the enhancing effect decreased again. NO caused a rapid and transient increase in intracellular guanosine-3',5'-cyclic monophosphate (cGMP) levels. The enhancing effect of NO on random migration was largely reversed by the inhibitors of cGMP accumulation, LY-83583 and methylene blue, and by the antagonists of cGMP-dependent protein kinase, 8-bromoguanosine-3',5'-cyclic monophosphorothioate, Rp-isomer (Rp-8-Br-cGMPS) and 8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphorothioate (Rp-8-pCPT-cGMPS). These observations strongly suggest that the enhancement of random migration by NO is mediated by cGMP and cGMP-dependent protein kinase. The effect of NO on migration did not occur in the absence of extracellular calcium. Although NO did not induce a measurable elevation of intracellular free calcium, pre-incubation with the intracellular calcium chelator Fura-2/AM abolished the enhancing effect of NO. It appears therefore that a small change in the level of cytoplasmic free calcium does play a role in the enhancement of random migration by NO. High concentrations of NO were found to inhibit chemotaxis induced by an optimal concentration of the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP). This inhibitory effect was also dependent on the presence of extracellular calcium. A role for cGMP in the inhibition of fMLP-induced chemotaxis by NO is not supported by our measurements of intracellular cGMP levels. In contrast to the effects on fMLP, NO did not affect chemotaxis induced by the phorbol ester PMA. In conclusion, we show that NO, not derived from NO donors but applied directly, may stimulate or inhibit neutrophil migration, dependent on the concentration. The enhancing effect of NO on random migration is mediated by cGMP, which emphasizes the importance of this second messenger as a modulator of neutrophil functional.

Calcium Spiking in Plant Root Hairs Responding to Rhizobium Nodulation Signals

Rhizobium lipochitooligosaccharide signal molecules stimulate multiple responses in legume host plants, including changes in host gene expression, cell growth, and mitoses leading to root nodule development. The basis for signal transduction in the plant is not known. We examined cytoplasmic free calcium in host root hairs using calcium-sensitive reporter dyes. Image analysis of injected dyes revealed localized periodic spikes in cytoplasmic calcium levels that ensued after a characteristic lag following signal application. Structural features of the signal molecules required to cause nodulation responses in alfalfa are also essential for stimulating calcium spiking. A nonnodulating alfalfa mutant is defective in calcium spiking, consistent with the possibility that this mutant is blocked in an early stage of nodulation signal perception.

Flow cytometric study of changes in the intracellular free calcium during the cell cycle.

We measured the intracellular levels of free cytoplasmic calcium in different phases of the cell cycle in viable rat fibroblasts, using two parameter flow cytometric analysis with Hoechst 33342 as the DNA specific dye and Fluo-3 as the calcium sensitive dye. We studied changes in calcium levels during the G1 phase of cell cycle by arresting cells with chemical agents such as staurosporine and hydroxyurea or by density dependent arrest of cell growth. We show that levels of calcium are lowest at the beginning of G1 phase but rise steadily with its progression and culminate at the G1/S border. Our results suggest that complex changes occur in calcium levels during the process of mitotic division. During progression of S and G2 phases, calcium levels decline and increase respectively. Our results offer a new methodology to estimate intracellular calcium levels in specific phases of the cell cycle. Based upon these results we propose a general scheme representing the changes in the intracellular calcium concentration during the progression of the cell cycle.

Oxidized low density lipoprotein inhibits platelet plasma membrane Ca 2+-ATPase

Oxidized low density lipoprotein (LDL) has been shown to enhance platelet activation. Since platelet activation is accompanied by an increase in cytoslic calcium, the effects of oxidized LDL on plasma membrane Ca 2+-ATPase plasma membrane fluidity and cytoplasmic calcium were studied in human platelets and purified platelet plasma membranes. Our results demonstrate that oxidized LDL, but not native LDL, inhibits the activity of Ca 2+-ATPase in purified platelet plasma membranes ( P < 0.01). Addition of the free radical scavenger α-tocopherol had no effect on the ability of oxidized LDL to inhibit the Ca 2+-ATPase. An increased cytoplasmic calcium level in whole platelets was induced by oxidized LDL ( P < 0.01), indicating that the plasma membrane Ca 2+-extrusion pump may also be inhibited in vivo by oxidized LDL, although other mechanisms for the increase in cytoplasmic calcium are possible. Since no change in membrane fluidity was observed in platelet plasma membranes exposed to oxidized or native LDL as estimated by steady state trimethylammonium diphenylhexatriene (TMA-DPH) anisotropy, oxidized LDL does not affect the Ca 2+-ATPase by grossly changing the membrane environment. The present results suggest that exposure of platelets to oxidized LDL causes inhibition of the plasma membrane Ca 2+-ATPase which contributes to the observed increase in cytoplasmic calcium and increased sensitivity to agonists.

Ca 2+ homeostasis and Ca 2+ signalling in sulphur mustard-exposed normal human epidermal keratinocytes

To investigate the role of increased cytoplasmic free calcium levels ([Ca] i) in sulphur mustard-induced cellular toxicity, the [Ca] i of human epidermal keratinocytes exposed to sulphur mustard concentrations between 50 and 800 μM were measured. No early increase in [Ca] i was observed during the first 20 min after application of the agent. Furthermore, at 3, 6 and 24 h after exposure to sulphur mustard, the [Ca] i of sulphur mustard-exposed cells differed only slightly from that of control cells. However, sulphur-mustard exposed cells responded with a weaker rise of [Ca] i upon stimulation with histamine and ATP than control cells. These results suggest that sulphur mustard can induce small increases in [Ca] i, which reflect abnormal cellular physiology rather than acute toxicity. In addition, it has been shown that sulphur mustard disturbs cellular Ca 2+ signalling in human epidermal keratinocytes.

Cytoplasmic Calcium Levels and Membrane Fluidity of Platelets in Contact with Polyether‐Polyamide Multiblock‐Copolymer Surfaces

Cytoplasmic calcium levels and the membrane fluidity of rabbit platelets stored in mini blood bags of crystalline-amorphous microstructured polymers (polyether-polyamide multiblock-copolymers) were studied. Fluorescent dye (Fura 2 or 1,6-diphenyl-1,3,5-hexatriene)-loaded platelet suspensions were stored at 37 degrees C for 1 h in the blood bags, and metabolic changes in the platelets during storage were evaluated by the fluorescent spectroscopic technique. The surfaces of poly(vinyl chloride) and polyolefin elastomers, which are used for commercially available blood bags, enhanced the progress of platelet metabolism; i.e., there was a dramatic decrease in membrane fluidity and an increase in [Ca2+]i. Furthermore, the decrease in membrane fluidity was observed prior to the increase in [Ca2+]i. These results suggest that the decrease in membrane fluidity of platelets in contact with polymer surfaces can be the dominant stage in the activation of these platelets. In contrast, the surfaces of polyether-polyamide multiblock-copolymers exhibited few changes in either membrane fluidity or [Ca2+]i levels. These results suggest that the platelets in contact with the crystalline-amorphous microstructured copolymer surfaces can be inert and inactivated in terms of the prevention of a decrease in membrane fluidity.